JP5156726B2 - Detection, identification and differentiation of eubacterial populations using hybridization assays - Google Patents

Abstract

The present invention relates to a method for the specific detection and/or identification of Staphylococcus species, in particular Staphylococcus aureus, using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region. The present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and/or identification of Staphylococcus species, in particular of S. aureus, in a biological sample. It relates also to nucleic acid primers to be used for the amplification of said spacer region of Staphylococcus species in a sample.

Description

  The present invention relates to the specific detection of Staphylococcus species, in particular Staphylococcus aureus, using a novel nucleic acid sequence derived from the ITS (spacer within the transcription region) region and / or Or it relates to a method for identification.

  The present invention also relates to a Staphylococcus species, particularly S. cerevisiae, in biological samples. The novel nucleic acid sequence derived from the ITS region between 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes to be used for the specific detection and / or identification of S. aureus.

  It also relates to nucleic acid primers to be used for amplification of the spacer region of Staphylococcus species in the sample.

  The genus Staphylococcus currently comprises 32 described species and 15 subspecies. From a human clinical point of view, Although S. aureus is the most important species, some coagulase negative species are episodic pathogens, especially in nosocomial infections among critically ill patients.

  Certain species of the genus Staphylococcus are isolated more frequently as the etiology of various infections in humans. The most important factor is S. S. aureus, S. S. epidermidis, S. epidermidis. S. haemolyticus, S. haemolyticus S. lug Dunensis, S. S. warneri and S. warneri. It is S. saprophyticus.

  S. S. schleiferi is considered an important pathogen in several European countries, but has been rarely reported in the United States, demonstrating the diversity of pathogens in the local epidemiology.

  In veterinary medicine, S. S. aureus, S. Intermedius and S. intermedius H. hyicus is the most prominent pathogen.

  Staphylococcus aureus is one of the most common nosocomial pathogens. It is responsible for several diseases ranging from epidermal abscesses to life-threatening intravascular infections. Its oral establishment that establishes long-term colonization of hospitalized patients and increased resistance to antibiotics makes it extremely difficult to control this organism in the hospital.

  S. between patients New knowledge on the epidemiology of S. aureus colonization reveals that it is potentially difficult to prevent nosocomial infections. S. in hospitals and communities. Effective control of S. aureus requires more aggressive means, including early diagnosis of prosthetic patients, in other words, a screening step.

  Since the frequency of Staphylococcus bacteremia is still increasing, it provides a faster method for detection and / or identification using more sensitive and more specific probes and / or primers It is necessary and urgent.

  The object of the present invention is that of Staphylococcus species, in particular S. cerevisiae. To provide a novel nucleic acid sequence derived from a specific region of the ITS of Staphylococcus species that can be used for detection and / or identification of S. aureus.

  Accordingly, the present invention provides an isolated nucleic acid molecule consisting of SEQ ID NO: 1, the RNA form of SEQ ID NO: 1 (where T is replaced by U), the complementary form of SEQ ID NO: 1, or any homologue And the use of said nucleic acid molecule as a target for the detection and / or identification of Staphylococcus species.

  Aspects of the present invention have as a target a specific region of the Staphylococcus aureus 16S-23S rRNA spacer region, which is a Staphylococcus species, in particular Staphylococcus aureus. aureus) for use as probes and / or primers allowing the detection and / or identification.

  Accordingly, the present invention relates to SEQ ID NO: 1 for the detection and / or identification of Staphylococcus species, in particular Staphylococcus aureus, or the RNA form of said SEQ ID NO: 1, wherein T is replaced by U), or an isolated nucleic acid molecule that specifically hybridizes to the complementary form of SEQ ID NO: 1, or any homologous sequence thereof, or a fragment of at least 20 contiguous nucleotides thereof.

  Another aspect of the invention relates to a set of probes for the detection and / or identification of Staphylococcus species in a sample, in particular Staphylococcus aureus.

  Another aspect of the present invention is a Staphylococcus species, in particular S. cerevisiae. It relates to a primer that allows specific amplification of the 16S-23S rRNA spacer region of S. aureus.

  An object of the invention is a composition containing any novel sequence of the invention, or any novel set of probes and / or primers of the invention, or a combination thereof.

  Another object of the invention is a kit in which said probes and / or primers are used for the detection and / or identification of Staphylococcus species, in particular Staphylococcus aureus. .

  Another object of the present invention is a rapid and reliable hybridization method for the detection and / or identification of Staphylococcus species, in particular Staphylococcus aureus.

  Another object of the present invention is a real-time PCR-based hybridization method for the detection and / or identification of Staphylococcus species, in particular Staphylococcus aureus.

Table Description Table 1: Sequence Listing.
Table 2: Primer pairs.
Table 3: Probe set.
Table 4: Staphylococcus species

  The following description serves to illustrate the terms and expressions used in the different embodiments of the invention described below.

  The terms “spacer” and “ITS” (spacers within the transcription region) are both abbreviated terms that refer to the region between 16S and 23S rRNA or between the 16S and 23S rRNA genes.

  The term “probe” refers to a single stranded oligonucleotide or polynucleotide having a sequence that is sufficiently complementary to hybridize to a target sequence to be detected.

  Preferably, the probes of the invention are more than 70%, 80%, 90% or 95% homologous to the exact complement of the target sequence to be detected. These target sequences are either genomic DNA or precursor RNA, or amplified versions thereof.

  The probes of the present invention excise the sequence from the cloned plasmid using an appropriate nuclease, if necessary, by cloning a recombinant plasmid containing an insert containing the corresponding nucleotide sequence, eg, molecular weight Can be formed by collecting them by fractionation according to

  The probes according to the invention can also be synthesized chemically, for example by the conventional phospho-triester method.

  As used herein, the term “complementary” nucleic acid means that the nucleic acid sequences can form a double helix that is completely base-paired to each other.

  The terms “polynucleic acid”, “nucleic acid”, and “polynucleotide” refer to either double-stranded or single-stranded cDNA or genomic DNA or RNA containing at least 5, 10, 20, 30, 40, or 50 contiguous nucleotides. Corresponding to. Polynucleic acids shorter than 100 nucleotides are referred to as “oligonucleotides”.

  They also refer to modified nucleotides, such as inosine, or nucleotides containing modified groups that do not substantially alter their hybridization characteristics.

  In the present invention, a single-stranded polynucleic acid sequence is always presented from the 5 'end to the 3' end.

  They can be used as is or in their complementary form, or in RNA form (where T is replaced by U).

  The term “closest neighbor” means a taxon that is known or expected to be most closely related for DNA homology and should be distinguished from the organism of interest.

  The expression “taxon-specific hybridization” or “taxon-specific probe” means that the probe hybridizes only to DNA or RNA derived from the taxon for which it was designed and hybridizes to DNA or RNA from other taxonomic groups. It means not.

  The term taxon can refer to a complete genus or a subgroup within a genus, a species or subtype within a species (subspecies, serotype, sequencer, biotype ...).

  The term “specific amplification” or “specific primer” refers to the fact that the primers only amplify spacer regions from those organisms for which they are designed and not spacer regions from other organisms. Point to.

  The term “sensitivity” refers to the number of false negatives, ie if one of the 100 strains to be detected is overlooked, the test is (100−1 / 100)% = 99% sensitive.

  The term “specificity” refers to the number of false positives, ie, for 100 strains detected, if 2 strains appear to belong to an organism for which the test has not been designed, the specificity of the test is ( 100-2 / 100)% = 98%.

  Oligonucleotides or polynucleotides selected as being “preferential” exhibit sensitivity and specificity of greater than 80%, preferably greater than 90% and most preferably greater than 95%.

The term “solid support” can refer to any substrate to which a polynucleotide probe can be attached, provided that it retains its hybridization characteristics, provided that the background level of hybridization is Stays low. Usually the solid substrate is a microtiter plate, a membrane (eg nylon or nitrocellulose) or a microsphere (bead). Prior to application to the membrane or immobilization, it may be convenient to modify the nucleic acid probe to facilitate immobilization or improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH ₂ groups, SH groups, carboxyl groups, or coupling with biotin, haptens or proteins.

The term “labeled” refers to the use of labeled nucleic acids. Labeling can be accomplished by using labeled nucleotides incorporated during the polymerization step of amplification as exemplified by Saiki et al. (1988) or Bej et al. (1990) or of labeled primers. It can be done by use or any other method known to those skilled in the art. Nature of the label, even or non-isotopic A isotopes (such as ^{32 P,} 35 ^S) (biotin, digoxigenin, fluorescent dyes, biotin, enzymes, etc.).

  The term “signal” refers to a series of electromagnetic waves (eg, fluorescence) or changes in current carrying information. The signal can be viewed directly or can be visualized and / or interpretable by different means or devices.

  A “sample” may be any biological material. This biological material can be taken directly from an infected human or animal, or after culture or enrichment, or from food, the environment, etc.

  The biological material may be, for example, any type of exudate, bronchial lavage, blood, skin tissue, biopsy, lymphocyte blood culture material, colony, and the like. The sample can be prepared or extracted according to any technique known in the art.

  Staphylococcus species that are clinically relevant in the sense of the present invention are S. cerevisiae. S. aureus, S. S. auricularis, S. S. capitis, S. S. caprae, S. et al. S. cohni, S. S. epidermidis, S. epidermidis. S. haemolyticus, S. haemolyticus S. hominis, S. S. lug Dunensis, S. S. pasturi, S. pasturi. S. saccharolyticus, S. S. saprophyticus, S. S. schleiferi, S. S. simulans, S. simulans Warneri and S. warneri. It is S. xylosus (Table 4).

  ITS is already known for several Staphylococcus species (WO 96/00298).

  In further studies, whole genome sequencing of different Staphylococcus species indicates that these organisms contain five ribosomal RNA operons in their genome.

  In particular, within the Staphylococcus sp. The S. aureus strain exhibits various spacer sequences even within one single isolate.

  These different ITSs are more than 16 types of sequences, and their length also varies from 300 to 550 base pairs.

  In order to solve the problems caused by this extremely high diversity, the present invention is based on all staphylococcus species, in particular all clinically relevant staphylococcus species, more particularly S. It provides a specific region of ITS that has been identified and scoped for its great advantage of providing a unique target sequence for the detection and / or identification of S. aureus.

  In fact, it has been found that the target sequences of the present invention are found in all types of spacers of all Staphylococcus species, in particular, all clinically relevant Staphylococcus species.

  This particular region of ITS, also called “target region” or “target sequence”, is a nucleic acid molecule consisting of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleic acid molecule homologous to SEQ ID NO: 1 or 2, their RNA It can be defined as a form (where T is replaced by U) or a complementary form thereof.

  The term “target sequence” includes all homologous sequences found in the ITS of any Staphylococcus species, hereinafter said homologous sequences are also referred to herein as “homologues”. The degree of homology is then higher than 75%, typically higher than 80% and even higher than 90%.

  In the system of the present invention, a “homologue” is then a sequence homologous to SEQ ID NO: 1 or 2 or any fragment thereof located in the ITS region of any Staphylococcus species, SEQ ID NO: 1 and 2 Are S. Derived from the S. aureus strain.

  Novel polynucleotides for use as probes and / or primers designed from the target sequences of the present invention for the detection and / or identification of Staphylococcus species are also an object of the present invention.

  In other words, an object of the present invention relates to novel polynucleotides for use as probes and / or primers that hybridize with the target sequences of the present invention for the detection and / or identification of Staphylococcus species. .

  In particular, the object of the present invention is that of Staphylococcus species, in particular S. cerevisiae. SEQ ID NO: 1 or 2 for detection and / or identification of S. aureus, or RNA form of said SEQ ID NO: 1 or 2 (where T is replaced by U), or said SEQ ID NO: 1 or 2 An isolated nucleic acid molecule that specifically hybridizes to its complementary form, or a fragment of at least 20 contiguous nucleotides thereof, or any homologue thereof.

  Suitable polynucleotide probes are about 5 to about 50 bases in length, more preferably between about 10 and about 25 nucleotides, and are sufficiently homologous to the target sequence.

  The polynucleotides of SEQ ID NOs: 1-70 and any homologues thereof can be used as probes.

  Suitable probes are the polynucleotides and homologues of SEQ ID NOs: 14, 16-23, 25-32, 35-42.

  Preferred primers of the invention are single stranded DNA polynucleotides that can act as starting points for the synthesis of the target sequences of the invention. The length and sequence of the primers of the present invention must be such that they are capable of priming extension product synthesis.

  Preferably, the primer of the present invention is about 5 to about 50 nucleotides in length, preferably about 15 to about 25. Its particular length and sequence should be selected depending on the conditions used, such as temperature and ionic strength.

  Preferred primers of the invention amplify the target sequence. In other words, preferred primers of the invention amplify SEQ ID NO: 1 or SEQ ID NO: 2 and / or homologues.

  Preferred primers of the present invention are SEQ ID NOs: 51, 52, 53, 55, 58, 65, 67, 68, 69, 70, and homologues.

  The fact that an amplification primer does not necessarily have to exactly match the corresponding template sequence to ensure proper amplification is described in detail in the reference (Kwok et al., 1990).

  The amplification methods used are the polymerase chain reaction (PCR, Saiki et al., 1988), ligase chain reaction (LCR, Landgren et al., 1988; Wu & Wallace, 1989; Barrani (Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS, Kwoh et al., 1989), strand displacement Amplification (SDA, Duck, 1990; Walker et al., 1992) or amplification by Qβ replicase (Lizardi et al., 1988; Lomeli et al., 1989) or in the art May be any other any appropriate method for amplifying a known nucleic acid molecule Te.

  Suitable polynucleotides of the invention for use as primers or probes are listed in Table 1.

  The polynucleotides of the present invention may comprise the addition or removal of one or several nucleotides at any of their representative ends, or the alteration of one or more nucleotides within the sequence, or a combination of both Any may be different in sequence from any of the polynucleotides identified in Table 1, or their homologues, provided that the resulting equivalent is still targeted as the corresponding unmodified polynucleotide. Hybridizes with the sequence. Said equivalent polynucleotide shares at least 75% homology with the corresponding unmodified polynucleotide, preferably more than 80%, more preferably more than 85%.

  When using polynucleotide equivalents, hybridization conditions may need to be modified to obtain the same specificity as the corresponding unmodified polynucleotide.

  As a result, if polynucleotides are to be used in sets under the same hybridization conditions, it may therefore be necessary to modify the sequence of other polynucleotides. These modifications are described in, for example, Hames B and Higgins S (ed.): Nucleic acid hybridization. Practical approach. It can be carried out according to principles known in the art such as IRL Press (Press), Oxford, United Kingdom (UK), 1985.

  The polynucleotide primers and / or probes of the present invention are also phosphorothioates (Matsukura et al., 1987), alkyl phosphorothioates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen). ) Et al., 1993), or may contain intercalating agents (Asseline et al., 1984) and the like.

  Modified primers or probes require flexibility with respect to the conditions under which they are used to obtain the required specificity and sensitivity. However, the hybridization results should still be essentially the same as those obtained with unmodified polynucleotides.

  The introduction of these modifications may be advantageous to affect several characteristics such as hybridization kinetics, reversibility of hybridization, and biological stability of the polynucleotide molecule.

  The probes and primers of the present invention may comprise Staphylococcus species, in particular S. cerevisiae. The method of the present invention for the detection and / or identification of S. aureus and also used for purposes.

  Detection and / or identification of the target sequence can be carried out by using electrophoresis methods, hybridization methods or sequencing methods.

The method of the present invention for the detection of one or more Staphylococcus species in a sample comprises the following steps.
First, if necessary, the nucleic acid present in the sample is made available for amplification and / or hybridization.
Second, and if necessary, if nucleic acid is present, amplify with one or another target amplification system, as specified below. Usually, amplification is necessary to enhance subsequent hybridization signals. However, for some samples, or some sensitive signal amplification systems, amplification may not be necessary.
Third, the nucleic acid present in the sample or the resulting amplified product is contacted with a probe to allow hybridization to proceed.
Finally, detect hybrids using a simple and compatible detection system. The presence or absence of one or several staphylococcus species can be inferred from the observed hybridization signal or pattern.

  The amplification system used can be more or less universal depending on the specific application required.

  By using universal primers located in the conserved flanking regions (16S and 23S genes) of the rRNA spacer, the spacer region of most if not all organisms from enterococci are amplified.

  In some applications, it may be appropriate to amplify one or several Staphylococcus species, but not all organisms present in the sample. This can be accomplished using specific primers located in the target region of Staphylococcus species, for example, using the polynucleotides of SEQ ID NOs: 69 and 70 or their homologs as primer pairs as they are, Or preferably, the polynucleotides of SEQ ID NOs: 58 and 68 or homologues thereof may be used.

In particular, the method of the present invention for the detection and / or identification of Staphylococcus species in a sample, in particular Staphylococcus aureus, comprises:
(I) releasing, isolating and / or concentrating the polynucleic acid in the sample, if necessary;
(Ii) amplifying the 16S-23S rRNA spacer region, or a fragment comprising the target sequence, or the target sequence or fragment thereof, if necessary, with at least one suitable primer pair;
(Iii) the polynucleic acid of step (i) or (ii) and the target sequence (where the target sequence consists of SEQ ID NO: 1 or 2 or a homologue thereof), or their RNA form (where T is Hybridizing with at least one polynucleotide probe that hybridizes to a complementary form thereof, or a fragment of at least 20 contiguous nucleotides thereof,
(Iv) detecting the formed hybrid; and (v) interpreting the resulting signal, inferring the presence of Staphylococcus species, and / or identifying the Staphylococcus species in the sample. Identifying,
Comprising.

  Preferably, the probes of the invention hybridize under conditions of high stringency.

  Under high stringency conditions, only complementary nucleic acid hybrids are formed. Thus, the stringency of the assay conditions determines the amount of complementarity required between the two nucleic acid strands forming the hybrid. Stringency is selected to maximize the difference in stability between the hybrids formed by the target and non-target nucleic acids.

  The hybridization conditions are such that the hybridization signal obtained when the polynucleotide of the present invention specifically hybridizes to the target sequence is the signal obtained when the polynucleotide hybridizes to the target sequence in a non-specific manner. Is selected in a different way.

  In practice, for example, due to specific hybridization to the target, the intensity of the signal is 2, 5, 10 times or more intense (eg LiPA system) compared to non-specific hybridization to the target sequence Different signals can be visualized.

  Different signals can be visualized when different peaks are drawn in the melting curve analysis, for example also when using real-time PCR methods.

  The fragment referred to in the amplification or hybridization step of any method of the invention may comprise 20-50, 20-80 or 20-100 contiguous nucleotides of SEQ ID NO: 1 or 2 or any homologue.

  In one embodiment, a very simple and advantageous technique for the detection of target sequences potentially present in a sample is real-time PCR.

There are different formats for the detection of amplified DNA, notably TaqMan ^™ probes, Molecular Beacons probes, FRET hybridization probes.

For the TaqMan ^TM probe, the single stranded hybridization probe is labeled with two components. In accordance with the principle of fluorescence resonance energy transfer, when the first component, so-called fluorescent agent, is excited with light of an appropriate wavelength, the absorbed energy is transferred to the second component, so-called quencher. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is digested during the extension phase by the 5'-3 'exonuclease activity of a polymerase, eg, Taq Polymerase. As a result, the excited fluorescent component and the quencher are spatially separated from each other and can therefore measure the fluorescence emission of the first component (EP B 0 543 942 and US Pat. No. 5,210,015).

  With respect to Molecular Beacons probes, the probes are also labeled with a first component and a quencher, and the labels are preferably located at different ends of the probes that are at least partially self-complementary. As a result of the secondary structure of the probe, both components are in spatial proximity in solution. After hybridization with the target nucleic acid, both components are separated from each other so that the fluorescence emission of the first component can be measured after excitation with light of the appropriate wavelength (US Pat. No. 5,118,801). Issue description).

  The fluorescence resonance energy transfer (FRET) hybridization probe test format is particularly useful for all types of homologous hybridization assays (Matthews, JA) and Clickica, LJ. Kricka, LJ), Anal Biochem 169 (1988) 1-25). It is used simultaneously and features two single-stranded hybridization probes that are complementary to adjacent sites on the same strand of the target nucleic acid (amplified). Both probes are labeled with different fluorescent components. When excited with light of the appropriate wavelength, the fluorescence resonance energy is such that the second component can only be measured if both hybridization probes bind to adjacent positions of the target molecule to be detected. According to the principle of movement, the first component transfers the absorbed energy to the second component.

  When annealed to the target sequence, the hybridization probes must be located very close to each other in head-to-tail alignment. Usually, the gap between the labeled 3 ′ end of the first probe and the labeled 5 ′ end of the second probe is as small as possible, especially about 0 to 25 bases, preferably about 1 to about 5 bases. It becomes more. This allows close access of FRET donor and FRET acceptor compounds, typically 10-100 angstroms.

  Alternatively, the decrease in fluorescence of the FRET donor compound can be monitored as a quantitative measure of the hybridization event in order to monitor the increase in fluorescence of the FRET acceptor compound.

  Among all detection formats known in the field of real-time PCR, the FRET-hybridization probe format has proven to be highly sensitive, accurate and reliable (WO 97/46707, WO 97 / 46712 pamphlet, WO 97/46714 pamphlet). It should be noted that the design of appropriate FRET hybridization probe sequences can sometimes be limited by the spatial characteristics of the target nucleic acid sequence to be detected.

  As an alternative to the use of two FRET hybridization probes, it is also possible to use fluorescently labeled primers and a single labeled polynucleotide probe (Bernard, PS et al. Anal.Biochem.255 (1998) 101-7). In this regard, it can be arbitrarily chosen whether the primer is labeled with a FRET donor or a FRET acceptor compound.

  For melting curve analysis, a FRET hybridization probe (Hyprobe or FRET-probe) can also be used (WO 97/46707, WO 97/46712, WO 97/46712). 97/46714 pamphlet). In such an assay, the target nucleic acid is first amplified in a typical PCR reaction with appropriate amplification primers. Hybridization probes can always be present during the amplification reaction or can be added thereafter. After completion of the PCR reaction, the temperature of the sample increases continuously. Fluorescence is detected as long as the hybridization probe binds to the target DNA. At the melting temperature, the hybridization probes are released from their targets and the fluorescent signal immediately decreases to the background level. This decrease is monitored with an appropriate fluorescence vs. temperature-time plot so that a negative value of the first derivative function can be calculated. The temperature value corresponding to the maximum value obtained for such a function is then taken as the determined melting temperature of said pair of FRET hybridization probes.

  Point mutations or polymorphisms within the target nucleic acid result in less than 100% complementarity between the target nucleic acid and the FRET probe, thus resulting in a decrease in melting temperature. This allows for common detection of pools of sequence variants by FRET-Hyprobe hybridization, while different members of the pool will subsequently be identified by performing a melting curve analysis. Can be.

  Instead of FRET hybridization probes, Molecular Beacons may alternatively be used for melting curve analysis.

When real-time PCR and homologous real-time PCR melting curve analysis are available, double-stranded DNA binding dyes such as SybrGreen ^™ I, or alternatively hybridize to different but similar target sequences Using any of the hybridization probes specifically designed to enable the identification of specific types of species or strains.

  In the first case, the melting temperature of the generated double-stranded PCR product must be determined. It should be noted that this method has only limited use because small differences in melting temperature result in only a small difference in melting temperature, and small differences cannot be monitored efficiently.

  Alternatively, the hybridization probe may be used in such a way that the melting temperature of the probe / target nucleic acid hybrid is determined.

There are different real-time PCR platforms such as ABI / Prism ^TM equipment, especially the LightCycler ^TM instrument, all of which measure light emission, continuously monitor emission peaks during the melting cycle, label Based on the same principle consisting of determining and visualizing the temperature (melting peak) at which the probe is separated from the amplification product. Mismatch between probe and target affects melting kinetics, resulting in a different melting peak for each species of interest, so melting peak data is characteristic of a specific [probe: target] sequence. It is.

The LightCycler ^™ platform has many advantages, especially time-saving and hybridization probes (HybProbe), TaqMan ^™ probes, Molecular Beacons and biprobes. Grants possible use of several different sequence specific fluorescent probe detection systems such as (SYBR Green I).

  In a preferred method of the present invention, two adjacent polys derived from the target region of the present invention are spaced in the head-to-tail orientation by a few nucleotides, generally 0-25, preferably about 1 to about 5. A hive probe system consisting of nucleotide probes is used. One of the probes is labeled at the 3 'end with a donor dye, the other is labeled with an acceptor molecule at the 5' end and is phosphate blocked at the 3 'end (to prevent acting as a primer). The donor dye is generally fluorescein and the acceptor molecule is generally LC Red 640 or 705.

  Detection of the target sequence of the present invention can also be achieved by an internal labeled PCR strand and a detection probe located on the opposing strand. The signal depends on the spatial proximity of the dye and depends on the amount of target.

  When both probes are hybridized to their target sequence, the emitted light of the donor is transferred to the acceptor fluorophore by fluorescence resonance energy transfer (FRET), and the emitted fluorescence (640 or 705 nm) is detected. Can do. The intensity of emitted fluorescence increases in parallel with the target DNA, the product of amplification.

The LightCycler probe is advantageous over the TaqMan ^™ probe in that it does not require hydrolysis and therefore does not require further extension of the PCR time (annealing-extension ≦ 12 seconds). Therefore, it is possible to complete the PCR program in just 45 minutes using the LightCycler fast thermal cycle.

  And the most recent generation of this real-time PCR platform is able to monitor several probes in a single reaction, detecting different staphylococci at species and also lower taxonomic levels And / or enables identification.

  Furthermore, it has been shown that a method designed for TaqMan technology can be easily converted to HybProbe technology with equivalent results (Haematologica 85 (12) 1248- 1254, December 2000).

  Accordingly, another object of the present invention relates to a set of at least two polynucleotide probes, also referred to as hive probes (HybProbe), both of which are between the two hive probes (HybProbe). It hybridizes to the same target sequence adjacent to each other by nucleotides, preferably 10 nucleotides or less, particularly 5 nucleotides or less.

  Hybridization of the target sequence with two hive probes (HybProbe) results in one hive probe such that the donor and acceptor fluorophores are within 0-25 nucleotides of each other, preferably within 0-5 nucleotides of each other. (HyProbe) is labeled with an acceptor fluorophore and the other is labeled with a donor fluorophore of a fluorescence energy transfer pair.

  To detect and / or identify Staphylococcus species, in particular clinically relevant Staphylococcus species, a set of two polynucleotide probes can be used, said two probes Hybridizes to SEQ ID NO: 1 or SEQ ID NO: 2, or the RNA form of said SEQ ID NO: 1 or 2 (where T is replaced by U), or the complementary form of SEQ ID NO: 1 or 2, or the complement Here, there are 25 or fewer nucleotides, preferably 5 or fewer nucleotides between the two probes.

  The probe set of the present invention also consists of 3, 4, 5, 6, 7, 8, 9, 10, or more probes, but preferably it comprises 2 to 5 probes, more preferably 2 or 3 It consists of two probes.

  The probe sets listed in Table 3 and their homologues are the preferred set of the present invention.

  Two polynucleotide sets (one for use as a primer and the other for use as a probe) can also be used, both of the primer and probe being SEQ ID NO: 1 or SEQ ID NO: 2, hybridizes to the RNA form of the SEQ ID NO: 1 or 2 (where T is replaced by U), the complementary form of the SEQ ID NO: 1 or 2 or a target sequence consisting of the complement, wherein the primer And no more than 25 nucleotides, preferably no more than 5 nucleotides, between said probes.

  The set of at least two polynucleotides of the present invention comprises a Staphylococcus species, in particular S. cerevisiae. Used in methods for detection and / or identification of S. aureus.

The method of the present invention for the detection and / or identification of Staphylococcus species in a sample comprises:
(I) releasing, isolating and / or concentrating the polynucleic acid in the sample, if necessary;
(Ii) amplifying the 16S-23S rRNA spacer region, or the target sequence, or part of the spacer comprising the target sequence, or part of the target sequence with at least one suitable primer pair;
(Iii) hybridizing a polynucleic acid and at least one set of at least two hive probes (HybProbe) that hybridize to the target sequence, wherein the target sequence is SEQ ID NO: 1 or 2, or An RNA form of SEQ ID NO, wherein T is replaced by U, or a complementary form of said SEQ ID NO, or any complement, or a fragment of at least 20 contiguous nucleotides thereof,
(Iv) detecting the hybrid formed in step (iii);
(V) inferring the presence of a Staphylococcus species from the unique hybridization signal obtained in step (iv) or identifying a Staphylococcus species in a sample;
Comprising.

  For example, the primer pair used in the amplification step is a forward primer consisting of SEQ ID NOs: 45, 49, 50, 52, 56, 61, 63, 64, 65, 66, 67, 68 or homologues thereof, and a sequence Any combination of reverse primers consisting of the numbers 46, 47, 48, 51, 53, 54, 55, 57, 58, 59, 60, 62 or homologues thereof.

  For example, the set of two hive probes (HyProbe) used in the hybridization step is any combination of two hive probes (HyProbe) selected from among polynucleotides of SEQ ID NOs: 3-70 or homologues thereof. However, the gap between the two hive probes (HybProbe) when hybridizing to the target sequence is less than 25 nucleotides, preferably less than 5 nucleotides.

  One advantage of the Hive Probe system is the fact that it allows detection of sequence variations, including mutations, polymorphisms and other mutant nucleic acid species, based on the following molecular concepts: . One of the hive probes (HybProbe) is a tightly bound “anchor probe”, while the adjacent “sensor probe” spans the region of sequence variation. During melting of the final PCR product, sequence changes are detected as changes in the melting temperature (Tm) of the sensor probe.

  For example, if the sample contains only SEQ ID NO: 1, a single melting peak is created using a hive probe (HyProbe) that specifically hybridizes to the SEQ ID NO: 1. If homologues are also present in the sample, in general, by using the same two hive probes (HybProbe) as long as there is one mismatched base that induces a temperature shift that can be easily observed, 2 Two peaks are created.

  Depending on the selected polynucleotides, their Tm and hybridization conditions, fluorescence can be measured during the amplification step, and then an amplification curve is generated or, after the amplification step, for melting curve analysis Create a melting curve.

  Thus, the resulting signal can be visualized in the form of an amplification curve or in the form of a melting curve, from which it is possible to infer the presence of a Staphylococcus species and / or which of Staphylococci It is possible to infer whether a species exists.

In particular, methods for the detection and / or identification of Staphylococcus species in a sample also include:
(I) releasing, isolating and / or concentrating the polynucleic acid in the sample, if necessary;
(Ii) amplifying the target sequence, or part thereof, with a labeled primer pair;
(Iii) a polynucleic acid and SEQ ID NO: 1, or the RNA form of SEQ ID NO: 1, wherein T is replaced by U, adjacent to the labeled primer with less than 25 nucleotides in between, or Hybridizing with a complementary form of SEQ ID NO: 1, or any complement, or at least one hive probe that hybridizes to a fragment of at least 20 contiguous nucleotides;
(Iv) detecting the formed hybrid;
(V) inferring the presence of a Staphylococcus species from the signal obtained in step (iv) and / or identifying the Staphylococcus species in the sample;
Comprising.

  The method of the present invention using the HybProbe system is adapted for the detection and identification of Staphylococcus aureus, and S. cerevisiae from other species. It is possible to distinguish between S. aureus, in particular coagulase negative Staphylococci (CoNS).

  Then, in the amplification step, suitable primers specifically amplify the target sequence consisting of SEQ ID NO: 1, or the RNA form of said SEQ ID NO (where T is replaced by U), or the complementary form of said SEQ ID NO A primer pair.

  In the hybridization step, the hive probe (HybProbe) should specifically hybridize to SEQ ID NO: 1 or 2, or the RNA form (where T is replaced by U), or the complementary form.

  Therefore, S. The S. aureus strain can be clearly distinguished from all other organisms tested by melting curve analysis.

  Furthermore, only CoNS gives rise to melting peaks, and unrelated signals are obtained with non-staphylococci or human genomic DNA.

  The preferred primer pair used in this particular example is selected between the forward primer selected between SEQ ID NO: 68 or 69 or homologues thereof and SEQ ID NO: 58 or 70 or homologues thereof. Any combination of reverse primers.

  The set of hive probes listed in Table 3 (HybProbe) or their homologues is a preferred set of hive probes of the present invention (HybProbe). A more preferred set of two hive probes (HybProbe) consists of SEQ ID NO: 17 or homologue and SEQ ID NO: 19 or homologue.

  The set of hive probes (HybProbe) consisting of SEQ ID NOs: 17 and 19 is highly sensitive and has S. S. aureus, S. S. epidermidis, and S. epidermidis It is possible to detect S. haemolyticus.

  Each polynucleotide listed in Table 1 corresponding to SEQ ID NO: 1 to SEQ ID NO: 70 and any homologues thereof may be used alone or in combination in any method of the present invention as a primer and / or probe. it can.

  A second embodiment based also on hybridization methods is the Line Probe Assay technology. The Line Probe Assay (LiPA) is a reverse hybridization that uses membrane strips to which several polynucleotide probes (including negative or positive control polynucleotides) can be conveniently applied as parallel lines. The format (Saiki et al., 1989).

  The LiPA technology described in Stuyver et al. (1993) and International Application WO 94/12670 provides a rapid and easy-to-use hybridization test. The result can be read within 4 hours after the start of amplification. Usually, after amplification in which the non-isotopic label is incorporated into the amplified product, and alkaline denaturation, the amplified product is contacted with the probe on the membrane and allowed to hybridize for about 1 to 1.5 hours. Thus, the hybrid formed is detected by enzymatic procedures, resulting in a visible purple brown precipitate. The LiPA format is fully compatible with commercially available scanning devices and thus allows automatic interpretation of results. All these advantages make the LiPA format reliable for use in everyday settings.

  The LiPA format is an advantageous tool for the detection and / or identification of pathogens at the species level, but also at higher or lower taxonomic levels. For example, the probe geometry for LiPA strips allows them to detect a complete genus of Staphylococcus or species within the genus (eg, Staphylococcus aureus), and Epidermidis, etc.) can be identified, or in some cases, subtypes within species (subspecies, serotypes, sequencers, biotypes, etc., anything clinically relevant) You can choose in a way that you can.

  The ability to simultaneously generate hybridization results with multiple probes is another benefit of LiPA technology. In many cases, the amount of information that can be obtained by a particular combination of probes is significantly greater than the data obtained by using a single probe assay. Therefore, the selection of probes for membrane strips is most important because an optimized set of probes allows for maximum information.

  These probes can be applied to the membrane strip at different locations and if at least one of these probes is positive, the result is interpreted as positive. Alternatively, these probes can be applied as a mixture at the same location, thereby reducing the number of lines on the strip. Such a reduction can be simple in order to make the strip more concise or allow the total number of probes on one strip to be expanded.

  In terms of practical benefits, another alternative approach is then a sequence of two or more different probes, called denatured probes, that can be further processed and applied to the strip as a single polynucleotide molecule. Synthesis of a polynucleotide having This approach considerably simplifies the LiPA-strip manufacturing procedure. For example, probes with nucleotide sequences A and B both need to detect all strains of Taxon X. In the latter alternative, a probe having the nucleotide sequence AB can be synthesized. This probe has the combined characteristics of probes A and B.

  Due to the properties mentioned above, the LiPA system can be regarded as an efficient format for the hybridization method, where several organisms need to be detected in the sample simultaneously.

  However, in any other hybridization assay, it should be clear that different probes can be used under the same hybridization and washing conditions for the detection and / or selection methods mentioned above can be used. It is. For example, it may be possible to immobilize the target nucleic acid on a solid support and use a mixture of different probes, all labeled separately, resulting in a different detection signal for each probe hybridized to the target . And now many different supports are available.

As an example, the procedure to be followed for the detection of one or more Staphylococcus species in a sample using the LiPA format is outlined below.
First, if necessary, the nucleic acid present in the sample is made available for amplification and / or hybridization.
Second, if present, the nucleic acid is amplified with one or another target amplification system, as described below. Usually, amplification is necessary to enhance subsequent hybridization signals.
Third, ultimately, after the denaturation step, the nucleic acid present in the sample or the resulting amplified product is converted into one or more probes (DNA-, RNA) that allow detection of the organism of interest. -Denatured or modified probe) is brought into contact with the immobilized LiPA strip to allow hybridization to proceed.
Finally, after the final washing step, detect the hybrid using a simple and compatible detection system. From the observed hybridization signal or pattern, the presence or absence of one or several organisms screened in a particular biological sample can be inferred.

  The amplification system used can be more or less universal depending on the specific application required.

  By using the conserved flanking regions of the rRNA spacer, ie universal primers located in the 16S and 23S genes, the spacer region of most but not all organisms from eubacteria are amplified.

  In some applications, it may be appropriate to more specifically amplify Staphylococcus species, although not all organisms present in the sample. This can be accomplished using specific primers located in the target region of Staphylococcus, eg, the polynucleotides of SEQ ID NOs: 69 and 70 or their homologs as such primer pairs. Can be used.

The method of the present invention for the detection and / or identification of Staphylococcus species in a sample comprises:
(I) if necessary, freeing, isolating and / or concentrating polynucleic acids present in the sample;
(Ii) amplifying the 16S-23S rRNA spacer region, or part thereof, with at least one suitable primer pair, if necessary;
(Iii) polynucleic acid and SEQ ID NO: 1 or 2, or the RNA form of said SEQ ID NO: 1 or 2 (where T is replaced by U), or the complementary form of said SEQ ID NO, or any homologue, or Hybridizing with at least one probe that hybridizes to a target sequence consisting of a fragment of at least 20 contiguous nucleotides;
(Iv) detecting the hybrid formed in step (iii);
(V) identifying microorganisms present in the sample from the different hybridization signals obtained in step (iv);
Comprising.

  The part of the ITS referred to in the amplification step can be a polynucleotide comprising the target sequence, or the target sequence itself, SEQ ID NO: 1 or 2, or the RNA form of said SEQ ID NO: 1 or 2 (where T is U Or a target sequence consisting of a complementary form of SEQ ID NO: 1 or 2, or any homologue, or a fragment of at least 20 contiguous nucleotides thereof.

  Preferably, the present invention provides the above method, wherein at least two microorganisms are detected simultaneously.

  The set of probes described in step (iii) comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or more probes of the present invention, or equivalents thereof.

  In a preferred embodiment of the invention, the probe set described in step (iii) comprises at least two probes.

  Preferred probes are polynucleotides of SEQ ID NOs: 1-70 and their homologues.

  The present invention also provides a method as described above, wherein the probe identified in step (iii) is present in a sample, preferably in combination with at least one other probe from the 16S-23S rRNA spacer region Allows simultaneous detection of different pathogenic bacteria

  Suitable probes are designed to achieve optimal performance under the same hybridization conditions such that they can be used in a set for simultaneous hybridization, which increases the utility of these probes. Highly increased, time and effort are gained significantly.

  Kits containing any polynucleotide of the present invention are also an object of the present invention.

The kit of the present invention comprises the following components:
A target sequence consisting of SEQ ID NO: 1 or 2, the RNA form of SEQ ID NO: 1 or 2 (where T is replaced by U), the complementary form of SEQ ID NO: 1 or 2, or a homologue thereof At least one polynucleotide that hybridizes to
A hybridization buffer or a component necessary to produce the buffer.

A suitable kit is
A target sequence consisting of SEQ ID NO: 1 or 2, which is adjacent to each other with less than 25 nucleotides, preferably less than 5 nucleotides, the RNA form of said SEQ ID NO: 1 or 2 (where T is replaced by U), said sequence At least one set of two hive probes (HybProbe) that hybridize to the complementary form of number 1 or 2, or any homologue thereof,
A hybridization buffer or a component necessary to produce the buffer.

  The methods used in the examples are Staphylococci, using a hive probe of SEQ ID NOs: 17 and 19 (HybProbe) in combination with a staphylococcus primer set of SEQ ID NOs: 58 and 68, in particular S. S. aureus, S. Epidermidis and S. epidermidis. A method for the detection of S. haemolyticus.

  The hive probe (HybProbe) is labeled with fluorescein on the 3 'side of SEQ ID NO: 17, and with LC red (LCred) 640 on the 5' side of SEQ ID NO: 19.

  In total, 63 strains of S. cerevisiae having different geographical origins. S. aureus isolate (including one representative of S. aureus subspecies anaerobic bacteria), 48 strains of S. aureus S. epidermidis isolate and 16 strains of S. epidermidis S. haemolyticus isolates were examined.

  T-RNA PCR (Bayneecoutte, M. et al., 1998, Int. J. Syst. Bacteriol. (48) 127-139) if the isolate did not give the expected results The gDNA was reclassified by and / or the culture was reclassified by ApiSTAPH (Biomelieux).

The instrument is a LightCycler ^™ (version 1.2) with appropriate software (LC-software, version 3.5) that allows real-time fluorescent PCR detection.

Example 1: Preparation of sample to be tested DNA from pure culture
Different purification methods can be used to prepare DNA from pure cultures.
Lysisafine (5 μg / μl) for 1 hour at 37 ° C. and purification with QIAamp blood DNA isolation kit (Qiagen) • Pitcher et al. Method (1989)
• MagNAPure LC DNA isolation kit III (bacteria, fungi) on MagNAPure instrument. Bacterial cells grown with O / N on LB plates or slants were suspended in 100-1000 μl TE pH 8 for storage at −20 ° C. 2 μl to 20 μl were used for extraction according to manufacturer's recommendations.
QIAamp DNA mini kit (catalog number 51306-QIAGEN). The culture was pretreated enzymatically using lysozyme and lysostaphin.

2 /. DNA from a positive blood culture bottle
Blood samples were seeded in aerated blood culture bottles (BacT / ALERT FA) and incubated in a BacT / Alert 3D system (Organon Teknika) at 37 ° C. until positive. Positives were monitored by a color change from dark green to yellow.

  Aliquots (1.5 ml) of blood cultures were frozen at −70 ° C. until use.

  Genomic DNA was prepared as described in the packaging insert of MPLC DNA isolation kit III. As recommended for Organon Teknika blood culture bottles, the eluate was centrifuged at 14000 rpm for 10 seconds to spin down extracted carbon particles before PCR.

Example 2: LightCycler (LC) Protocol Kit LC-FastStart DNA Master Master Catalog (Catalog No. 3003248 or 2239272) according to manufacturer's instructions
Any sample material suitable for PCR can be used for purification, concentration, and absence of inhibitors,
The primers should each be at a final concentration of 0.3-1 μM,
-Hive probe (HybProbe) at a final concentration of 0.2 μM or twice each,
The concentration of MgCl ₂ should be optimized and can vary from 1 to 5 mM,
• And the negative control should be activated.

  Amplification and melting conditions are described below. LC software version 3.5 was used. The quantitative setting was F2 / back F1 (sample). Arithmetic mode was used for baseline adjustment. The calculation of the crossing point (Ct) was based on the second derivative maximum. The calculation method of the melting peak was a polynomial method. The peak area was used to calculate Tm.

Example 3: Purified DNA, results of comprehensiveness and cross-reactivity tests 1 / Comprehensiveness S. aureus isolate (n = 63) was successfully amplified (Ct range 17.25-33.51) and 53.13 ° C. ± 0.52 ° C. for any geographical or species origin One homogeneous melting peak was obtained with an average Tm of.

  S. Subspecies S. aureus and S. aureus sp. It should be noted that S. aureus subspecies anaerobic bacteria also belong.

  S. S. epidermidis isolate (n = 48), and S. epidermidis All isolates accepted as S. haemolyticus (n = 16) could be detected using melting curves. A quantitative curve was not usually observed. S. The average Tm value of the S. epidermidis isolate is 44.55 ° C. ± 0.21 ° C. For S. haemolyticus, it was 44.96 ° C. ± 0.24 ° C. There were no differences between isolates from different geographic or species.

  S. S. aureus or S. aureus All isolates received as S. haemolyticus reacted as expected. However, five S.P. For S. epidermidis isolates, deviating results were obtained.

  Of these, two strains (one from the UK (UK) and one from Italy) were later Identified as S. hominis. Another S. cerevisiae from the UK S. epidermidis isolates exhibit a Tm value of 49.02 ° C. and are later identified as Staphylococcus species (S. aureus, S. epidermidis or S. epidermidis). .. can be determined as other than S. haemolyticus. The British isolate was later described in S. Reclassified as S. haemolyticus. And one isolate from Spain that produces a small melting peak that is not characteristic is S. cerevisiae. Intermedius or S. intermedius Reclassified as S. chromogenes.

2 /. Cross-reactivity More than 50 different bacterial species were tested (Mycobacterium, Pseudomonas, Streptococci, etc.) and some fungi were also tested. None of the tested organisms produced quantitative curves or melting peaks by assay.

3 /. result:
All investigated S.P. An S. aureus isolate was detected (100% sensitivity) and could be clearly distinguished from all other isolates studied.

  S. Epidermidis and S. epidermidis. Sensitivity was also 100% for S. haemolyticus.

  Thus, using this particular set of hive probes of SEQ ID NOs: 17 and 19 (HybProbe), both species are detected without discrimination from each other or from other CoNS.

  No unwanted cross-reactivity with the pathogen-derived organisms tested was observed.

Example 4: Blood Culture Results A total of 16 positive blood culture bottle aliquots seeded with patient samples were tested.

  S. Of the five bottles in which S. aureus grew, four could be identified as they were. One showed no peak. In fact, the sample DNA is S. Reclassified as S. schleiferi.

  S. Of the 10 bottles that were positive for S. epidermidis, 7 showed peaks between 44 ° C. and 45 ° C. (as expected), while 3 showed peaks shifted to 47 ° C. . After reclassifying the DNA of the deviating samples, they are Identified as S. hominis.

  S. One bottle that was positive for S. haemolyticus yielded the expected dissolution peak at 44.59 ° C.

Example 5: Other detections Epidermidis and S. epidermidis. A total of 51 CoNS strains were studied except for S. haemolyticus. The results are summarized in the following table.

Amplification was observed on the gel for all strains of all species except the two (S. schleiferi and S. scurii) whose results are ambiguous (below Also shown). Some CoNS produced growth curves (amplification) with corresponding Ct values, but the endpoint fluorescence was S. cerevisiae. It was very low (less than 0.004) compared to the value obtained for S. aureus (eg 0.03 for 10 ³ copies).

  Most species tested are S. cerevisiae. S. chromogenes, S. chromogenes. S. hyicus and S. With the exception of S. simulans, it was detectable by melting curve analysis.

  S. S. schleiferi and S. The results of S. scuri were unclear and melting peaks were observed for both species but could not be reliably distinguished from potential contaminants. The results obtained with the universal primer suggested that both species were probably not detectable and there was clear evidence on the gel, but the hive probe (HyProbe) produced no signal in the LC It was.

  The range of Tm observed for CoNS is 39-48 ° C. This is because S.A. Although clearly different from the case of S. aureus, S. aureus Epidermidis and S. epidermidis. Completely overlaps the range of S. haemolyticus.

Claims (5)

A set of two polynucleotide probes for detection and / or identification of Staphylococcus aureus , said two probes being SEQ ID NO: 1 or SEQ ID NO: 2 , or their RNA form (where T is Or is specifically hybridized to their complementary form, wherein there are no more than 25 nucleotides between the two probes , SEQ ID NOs: 15 and 20, or SEQ ID NOs: 15 and 21, Or a set of two polynucleotide probes consisting of SEQ ID NOs: 17 and 16, or SEQ ID NOs: 17 and 19, or SEQ ID NOs: 27 and 28, or SEQ ID NOs: 29 and 22, or SEQ ID NOs: 30 and 18 . A composition comprising a set of two polynucleotide probes according to claim 1 . (I) Samples and polynucleic acids from step for hybridizing a set of two polynucleotide probes according to at least one of claims 1,
(Ii) detecting the formed hybrid by performing a melting curve analysis ; and
(Iii) interprets the signals obtained, to infer the presence of Staphylococcus aureus, to identify Staphylococcus aureus in a sample step,
A method for the detection and / or identification of Staphylococcus aureus in a sample comprising. (I) releasing, isolating and / or concentrating the polynucleic acid in the sample;
(Ii) at least one suitable primer pair consisting of any combination of forward primer polynucleotides and reverse primer polynucleotides selected from the group consisting of SEQ ID NOs: 61 and 58 and SEQ ID NOs: 68 and 58 ; Amplifying the 23S rRNA spacer region, or a fragment comprising the target sequence, or the target sequence or fragment thereof;
(Iii) hybridizing the polynucleic acid of step (i) or (ii) with at least one set of two polynucleotide probes according to claim 1;
(Iv) detecting the formed hybrid by performing a melting curve analysis; and
(V) interpreting the resulting signal, inferring the presence of Staphylococcus aureus, and identifying Staphylococcus aureus in the sample;
A method for the detection and / or identification of Staphylococcus aureus in a sample comprising. The following ingredients:
- at least one charge two polynucleotide probes of the set according to claim 1,
A kit for the detection and / or identification of Staphylococcus aureus , comprising a hybridization buffer or components necessary to produce said buffer.